Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration

The influence of MgATP on the Ga⁺⁺-activated isometric tension of skinned frog muscle fibers was examined in solutions containing: Mg⁺⁺ = 5 mM, creatine phosphate (CP) = 14.5 mM, creatinephosphokinase (CPK) = 1 mg/ml, total EGTA = 7 mM, CaCl₂, KCl, imidazole ≥ 20 mM so that ionic strength = 0.15, pH = 7.00, and MgATP = 2 mM, 0.1 mM, or 20 µM. CP and CPK were necessary for these experiments as determined experimentally by their effect on the tension-Ca⁺⁺ relation, which was saturated for CP ≥ 14.5 mM. This was interpreted to mean that sufficient CP was present to effectively buffer MgATP intracellularly. Decreasing MgATP shifts the tension-pCa curve to higher pCa (-log Ca⁺⁺) so that, for half-maximal tension: pCa_1/2 = 4.5 for MgATP = 2 mM, pCa_1/2 = 5.1 for MgATP = 0.1 mM, and pCa_1/2 = 5.8 for MgATP = 20 µM; maximum isometric tension is the same in all cases, however. If MgATP was decreased to 1 µM, tension at Ga⁺⁺ > 10^–8 M was 84% of the maximum Ca^-+-activated tension in 2 mM MgATP and increased only slightly to 90% for pCa = 4.5. Weber (1970, In The Physiology and Biochemistry of Muscle as Food, Volume 2, E. J. Briskey, R. G. Cassens, and B. B. Marsh, University of Wisconsin Press, Madison, Wis.), using similar solutions, observed similar shifts in half-maximal calcium activation of rabbit myofibril ATPase rates. In explanation, Weber and Bremel (1971, In Contractility of Muscle Cells and Related Processes, R. J. Podolsky, editor, Prentice-Hall, Inc., Englewood Cliffs, N.J.; Bremel and Weber, 1972, Nat. New Biol., 238:97) have described a mechanism whereby, at low ATP, "rigor complexes" are formed between myosin and thin filament actin and, in turn, alter the calcium affinity of one class of the two Ca⁺⁺-binding sites on troponin, so that the thin filament is "turned on" for contraction at lower Ca⁺⁺ levels. Tension data from skinned fibers substantially supports this hypothesis. A stability constant for CaEGTA of 2.62 x 10¹⁰ M^–1 was determined, with the help of F. N. Briggs, in solutions similar to those used for skinned fibers and was the same for 100 and 300 mM KCl.     

Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits.     

The role of ATP in energy-deprivation contractures in unloaded rat ventricular myocytes

Energy-deprivation contractures were investigated in unloaded rat ventricular myocytes. Application of 2 mM cyanide in the presence of 10 mM 2-deoxyglucose (metabolic blockade) led to a rapid shortening "contracture" (maximum speed 1.5 +/- 0.2% control cell length/s). Cells shortened to a constant length of 69 +/- 1.6% of the control length. Removal of cyanide caused cells to shorten further ("recontracture"), before relaxing towards the control length. Cells shortened to 57 +/- 2.0% during the recontracture. Similar behaviour was observed in zero extracellular [Ca2+]. Cells permeabilized with saponin (0.1% w/v) responded to the removal of ATP from the bathing solution, and to readdition of ATP, as intact cells did to complete metabolic blockade and its removal. In these permeabilized cells, the extent and speed of contracture shortening were similar at pCa = 7 and pCa greater than 9. When the bath concentration of ATP ([ ATP]b) was lowered to zero, shortening stopped at about 70% of the control length. However, when [ATP]b was lowered to an intermediate level (4-20 microM), cells contracted to lengths as short as 30% of the control length. Similarly, when [ATP]b was restored from zero to an intermediate concentration (4-20 microM), recontracture shortening continued without relaxation. The peak speed of this Ca2(+)-independent shortening showed a sigmoidal dependence on pMgATP (pMgATP0.5 = 4.0). Phosphocreatine (10 mM) shifted the ATP dependence of Ca2(+)-independent shortening to lower [ATP]b (pMgATP0.5 = 5.0), suggesting that gradients of [ATP] could exist between the bath and the myofilaments. Ca2(+)-independent shortening was inhibited by the chemical phosphatase 2,3-butanedione monoxime (BDM), although BDM did not relax cells from the shortened state during energy deprivation. Using a simple model, we show that the results can be explained by cross-bridge cycling occurring independently of Ca2+ over a "window" range of [MgATP] (0.1-100 microM). Therefore, when [MgATP] falls, cross-bridge cycling occurs and the cell shortens. As [MgATP] falls to very low levels ([ MgATP] less than 1 microM), shortening ceases as the rate of cross-bridge cycling declines. Recontracture occurs on restoring ATP production, because stiffness falls and Ca2(+)-independent cross-bridge cycling initially increases. As [MgATP] rises above 100 microM, Ca2(+)-independent cross-bridge cycling ceases and the cell relaxes towards the control length. We conclude that energy-deprivation contractures, and recontractures, can result from changes in [MgATP] and do not necessarily require changes in [Ca2+]i.     

pH dependence of the effect of adenosine triphosphate and ethylenediaminetetraacetate on sodium and magnesium binding by cellular membrane fragments

The pH dependence of previously reported effects of adenosine triphosphate (ATP) and ethylenediaminetetraacetate (EDTA) on cation binding by rat liver microsomes was studied by an equilibration and washing procedure. Equilibration of microsomes in media containing 95 mM NaCl and 4 mM MgCl₂ with pH varied from 4 to 8 resulted in an increase in bound cations from zero below pH 4 to 0.90 mmoles Mg and 0.34 mmoles Na/g N at pH 8; the ratio of bound Na/bound Mg increased from 0.15 at pH 5 to 0.38 at pH 8. Addition of 5 mM EDTA to the equilibration media produced striking changes in cation binding such that bound Na/bound Mg increased from 0.30 at pH 5 to 3.90 at pH 7 and decreased to 3.55 at pH 8. In the presence of added 10 mM ATP, bound Na/bound Mg increased from 0.10 at pH 5 to a maximum of 0.80 at pH 7. The observed changes could generally be correlated with known mass law relationships, although the system containing added ATP was complicated considerably by the hydrolysis of ATP. Results demonstrate that environmental pH is an important factor in determining the effect of ATP and EDTA on the cation binding pattern of cellular membranes. Because hydrogen ion is a product of ATP hydrolysis as well as of other metabolic reactions, the described interactions may be of particular significance in the molecular mechanisms of ATP effects on cation binding and transport in living cells.     

Contraction of rabbit skinned skeletal muscle fibers at low levels of magnesium adenosine triphosphate

The contractile properties of skinned single fibers from rabbit psoas muscle were investigated under conditions of low MgATP and no Ca2+ (i.e., less than 10(-8) M). At 1 microM MgATP, fibers shortened at a maximum velocity of 660 +/- 420 A/half sarcomere/s (n = 9), compared with 34,000 A/half sarcomere/s measured during maximum Ca2+-activation at 1 mM MgATP (Moss, R. L., 1982. J. Muscle Res. Cell. Motil ., 3:295-311). The observed dependence of Vmax on pMgATP between 7.0 and 5.3 was similar to that of actomyosin ATPase measured previously by Weber, A., R. Herz , and I. Reiss (1969, Biochemistry, 8:2266-2270). Isometric tension was found to vary with pMgATP in a manner much like that reported by Reuben , J. P., P. W. Brandt, M. Berman , and H. Grundfest (J. Gen. Physiol. 1971. 57:385-407). A simple cross-bridge model was developed to simulate contractile behaviour at both high and low levels of MgATP. It was found that the pMgATP dependence of Vmax and ATPase could be successfully modeled if the rate of detachment of the cross-bridge was made proportional to the concentration of MgATP. In the model, the similar dependence of Vmax and ATPase on pMgATP was derived from the fact that in this range of pMgATP every pass of a cross-bridge by an actin site resulted in an attachment-detachment cycle, and every such cycle caused hydrolysis of one molecule of ATP.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.     

Stimulation of rat liver mitochondrial adenosine triphosphatase by anions.

The hydrolysis of MgATP by isolated rat liver mitochondrial ATPase (EC 3.6.1.3) at pH 8.0 was stimulated by various anions. The rate of hydrolysis was increased from 18 to 170 mumol per min per mg, a 9.4-fold stimulation, by HSeO3 at 1 mM MgATP. In the absence of a stimulatory anion, reciprocal plots of initial velocity studies with MgATP as the variable substrate were curved (Hill coefficient approximately 0.5). With the addition of anion, the reciprocal plots became linear. When the substrate was MgITP or MgGTP with the isolated enzyme or MgATP with submitochondrial particles, no curvature of the reciprocal plots was observed. With purified ATPase, anions stimulated the hydrolysis of MgITP, MgGTP, MgUTP or MgCTP only slightly. With submitochondrial particles the stimulation by anions of MgATP hydrolysis was limited to approximately 2-fold. These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.     

Discrimination of assembled and disassembled forms of gizzard myosin by papain

Chicken gizzard myosin in 0.15 M or 0.5 M NaCl was cleaved at two sites of heavy chain with 2-10 micrograms/ml papain. MgATP inhibited these cleavages of myosin in 0.15 M NaCl but not in 0.5 M NaCl. The protective effect of ATP was observed at concentrations as low as 10 microM and increased in proportion to ATP concentration to a maximum at 1 mM. ADP was as effective as ATP, while adenosine 5'-[beta, gamma-imido]triphosphate, an unhydrolyzable ATP analogue, was less effective than ATP or ADP. AMP had no protective effect on the digestion of myosin and GTP inhibited slightly the digestion. When the papain-insensitive myosin in 0.15 M NaCl and 2.5 mM MgATP was phosphorylated by Ca2+/calmodulin-dependent myosin light-chain kinase, the myosin restored the vulnerability to papain. However, the two papain-susceptible forms, nonphosphorylated form in the absence of MgATP and phosphorylated form in the presence of MgATP, yielded very similar but distinct proteolytic fragments upon the digestion. When the extent of myosin assembly was estimated by the turbidimetry of myosin suspension in 0.15 M NaCl, nonphosphorylated myosin in the absence and presence of MgATP was assembled and disassembled, respectively, and phosphorylated myosin in the presence of MgATP was assembled. These results suggest that, at physiological ionic strength, papain as a probe distinguishes disassembled myosin and assembled myosin as papain-insensitive and papain-sensitive forms, respectively.     

Quantitative analysis of sea urchin egg kinesin-driven microtubule motility

We have analyzed the effects of various substrates and inhibitors on the rates of microtubule (MT) motility induced by sea urchin egg kinesin using real-time computer analysis and video-enhanced light microscopy. In the presence of magnesium, 10 mM concentrations of all the nucleotides tested supported MT translocation, with velocities in MgATP greater than MgGTP greater than MgTTP approximately equal to MgUTP greater than MgCTP greater than MgITP. The velocity of kinesin-driven MT motility is fairly uniform over approximately 3 pH units, from pH 6 to 9, with almost no motility outside this range. In the presence of ATP, no motility is observed in the absence of divalent cations; addition of Mg2+ but not addition of Ca2+ restores motility. MgATP-dependent MT motility is reversibly inhibited by Mg-free ATP, EDTA, or tripolyphosphate, suggesting that Mg-free ATP is an inactive substrate analogue. MgATP and MgGTP both obey saturable, Michaelis-Menten kinetics, with apparent Km values of approximately 60 microM and 2 mM, and Vmax values of approximately 0.6 and 0.4 microns/s, respectively. MgATP gamma S and MgADP are classic competitive inhibitors of kinesin-driven motility in MgATP, with Ki values of approximately 15 and 150 microM, respectively. Adenosine 5'-(beta, gamma-methylene)-triphosphate and N-ethylmaleimide only inhibit MT motility weakly, while adenyl-5'-yl imidodiphosphate and vanadate strongly inhibit MT motility, but not in a simple competitive manner. Moreover, in contrast to other inhibitors which cause a unimodal decrease in MT mean velocity, vanadate concentrations greater than approximately 10% that of MgATP cause some MTs to become immotile, resulting in a bimodal distribution of MT velocities.     

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.     

Reasons causing a lag period in the oxidative phosphorylation process. Isn't ATP an internal uncoupler of ATP synthetase?]

The kinetics of oxidative phosphorylation catalyzed by bovine heart submitochondrial particles was studied in a range of MgATP and MgADP concentrations from 0.3 to 10 mM. It is shown that, at a low uncoupler concentration (0.9 microM of tetrachlorotrifluoromethylbenzimidazole, the lag period of the reaction increases from 12 s to 2-3 min, and KM for Pi increases severalfold; the value of Vmax remains practically unchanged. Increasing the [MgATP]/[MgADP] concentration ratio, with their total concentration being unchanged, leads to similar changes in the kinetics of oxidative phosphorylation. The value of delta pH generated on the membrane of AS particles at delta microH+ = 60 delta pH was measured using 9-aminoacridine. It was found that the electrochemical potential of H+ ions shows the same thermodynamic shift in the reaction of energy-dependent Pi -ATP exchange throughout the [MgATP]/[MgADP] concentration range studied, from 0.1 to 10: the synthesis on the ATP molecule is provided by the transmembrane transfer of two H+ ions. It was shown that the binding of ATP and/or ADP in the allosteric site, whose saturation is necessary for the functioning of ATP synthase, occurs with equal constants, 1-2 mM. It is concluded that the lag period in the synthesis of ATP indicates the monomolecular transition ATP hydrolase-->ATP sysnthase, which comes about by the action of transmembrane potential. The binding of MgADP or MgATP renders the enzyme structure "more coupled" or "less coupled", respectively. Structural distinctions manifest themselves in a kinetically different behavior of mitochondrial ATP synthase at [MgATP] > [MgADP] and [MgATP] < [MgADP] and do not suggest futile leakage of H+ through the membrane.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Demembranated muscle fibers catalyze a more rapid exchange between phosphate and adenosine triphosphate than actomyosin subfragment 1

The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.     

The dependence of isometric tension, isometric ATPase activity, and shortening velocity of limulus muscle on the MgATP concentration.

The dependence of the isometric tension, the velocity of unloaded shortening, and the steady-state rate of MgATP hydrolysis on the MgATP concentration (range 0.01-5 mM MgATP) was studied in Ca-activated skinned Limulus muscle fibers. With increasing MgATP concentration the isometric tension increased to a peak at approximately 0.1 mM, and slightly decreased in the range up to 5 mM MgATP. The velocity of unloaded shortening depended on the MgATP concentration roughly according to the Michaelis-Menten law of saturation kinetics with a Michaelis-Menten constant Kv = 95 microM and a maximum shortening velocity of 0.07 muscle lengths s-1; the detachment rate of the cross-bridges during unloaded shortening was 24 s-1. The rate of MgATP splitting also depended hyperbolically on the MgATP concentration with a Michaelis-Menten constant Ka = 129 microM and a maximum turnover frequency of 0.5-1 s-1. The results are discussed in terms of a cross-bridge model based on a biochemical scheme of ATP hydrolysis by actin and myosin in solution.     

Inactivation of H+,K+-ATPase by a K+-competitive photoaffinity inhibitor

A light-sensitive derivative, 2,3-dimethyl-8-[(4-azidophenyl)methoxy]imidazo[1,2-a]pyridine (DAZIP), of the drug 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (SCH 28080) has been synthesized and shown to be a K+-competitive inhibitor of gastric H+,K+-ATPase in the dark. The apparent dissociation constants calculated for DAZIP at pH 6.4 and 7.4 were 1.8 +/- 0.2 and 4.7 +/- 1.2 microM, respectively. Inhibition required binding of DAZIP to a luminal-facing site on the enzyme. Irradiation in the presence of DAZIP and 2 mM Mg2+ resulted in irreversible loss of ATPase activity that was more than 2-fold greater at pH 6.4 than at pH 7.4, showing the enhanced efficiency of covalent incorporation at the lower pH. Further photolyses were conducted at pH 6.4 in the presence of either 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), ATP and CDTA, or MgATP. The specificity of light-dependent, covalent insertion of DAZIP for the site of reversible inhibition was shown both by protection against photoinactivation given by K+ (the competing ligand) and by the observation that the amount of K+-protectable photoinactivation approached a maximum limiting value as a function of DAZIP concentration. The effectiveness of K+ in protecting against photoinactivation was 100-fold greater in the presence of ATP and CDTA than in the presence of either Mg2+ or CDTA and suggests the formation of a ternary complex of the apoenzyme with ATP and tightly bound K+. The dissociation constant for DAZIP (2 microM) calculated from photolyses in the presence of MgATP without added K+ agreed with the kinetic experiments and suggests that DAZIP inhibits turnover by binding to E.MgATP.     

Ability of a phosphocreatine-myofibrillar creatine kinase system to prevent the rigor tension of myocardial fibers

In the calcium-free medium the EGTA-treated rat myocardial fibres developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and the maximal tension at 0.1 mM. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. In the presence of MgADP phosphocreatine decreased rigor tension more rapidly and to the higher extent than MgATP. At 5 mM MgADP half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the km value for phosphocreatine in the creatine kinase reaction. These results demonstrate that the native creatine kinase in the EGTA-treated fibres is able to create high local ATP concentration in the myofibrillar compartment at the expense of phosphocreatine under the conditions of deficiency or even absence of ATP. It appears that at the energy supply disturbances the myocardial contracture develops at least partially due to low activity of the myofibrillar creatine kinase because of phosphocreatine deficiency.     

Effects of cobalt, magnesium, and cadmium on contraction of rat soleus muscle.

The effects on isometric tension of three divalent ions that block calcium channels, magnesium, cobalt, and cadmium, were tested in small bundles of rat soleus fibers. Cobalt, at a concentration of 2 or 6 mM, reversibly depressed twitch and tetanic tension and the depression was much greater in solutions containing no added calcium ions. Magnesium caused much less depression of tension than cobalt. The depression of tension was not accompanied by membrane depolarization or a reduction in the amplitude of action potentials. A reduction caused by 6 mM cobalt in the amplitude of 40 or 80 mM potassium contractures was not accompanied by a comparable reduction in tension during 200 mM potassium contractures, and could be explained by a shift in the potassium contracture tension-voltage curve to more positive potentials (by +7 mV on average). Similar effects were not seen with 2 or 6 mM magnesium. At a concentration of 20 mM, both cobalt and magnesium depressed twitch and tetanic tension, cobalt having greater effect than magnesium. Both ions shifted the potassium contracture tension-voltage curve to the right by +5 to +10 mV, caused a small depression of maximum tension, and slowed the time course of potassium contractures. Cadmium (3 mM) depressed twitch, tetanic, and potassium contracture tension by more than 6 mM cobalt, but experiments were complicated by the gradual appearance of large contractures that became even larger, and sometimes oscillatory, when the solution containing cadmium was washed out. It was concluded that divalent cations affect both activation and inactivation of tension in a manner that cannot be completely explained by a change in surface charge.